Hematological Laboratory Test

Dr. Rafal Al-Saigh

MBChB, PhD, FIBMS Path
Specialist Pathologist
Complete blood count


ESR


Bleeding Tendency


Blood grouping

CBC- compl e te bl
Component of the CBC:

o od count
• Red Blood Cells (RBCs)
• Hematocrit (Hct)
• Hemoglobin (Hgb)
• Mean Corpuscular Volume (MCV)
• Mean Corpuscular Hemoglobin
Concentration
(MCHC)
- Red cell distribution width (RDW)
RDW-CV (Red cell distribution width-cell volume)
RDW-SD (Red cell distribution width-cell size)
• White Blood Cells (WBCs) (Di erential count)
• Platelet
 RBC
• RBC (varies with attitude):
– M: 4.7 to 6.1 x10^12 /L
– F: 3.8 to 5.4 x10^12 /L
• Biconcave disc shape with diameter
of about 8 µm
• Function: - transport hemoglobin which carries
oxygen from the lung to the tissues
Life span 100-120 days.
 ↓ in anemia
 ↑ in polycythemia
 Hemoglobin & Hematocrit
Hemoglobin :
M: 13.8 to 17.2 gm/dL
F: 12.1 to 15.1 gm/dL
Hematocrit : (packed cell volume) PCV
It is ratio of the volume of red cell to the volume of
whole blood.
M: 40.7 to 50.3 %
F: 36.1 to 44.3 %
PCV or Hematocrit

 57% Plasma
 1% Bu y coat – WBC
 42% Hct (PCV)
 MCV&MCHC
– MCV = mean corpuscular volume HCT/RBC
count= 80-100fL
• small = microcytic
• normal = normocytic
• large = macrocytic
– MCHC= mean corpuscular hemoglobin
concentration HB/RBC count= 26-34%
• decreased = hypochromic
• normal = normochromic
 MCH & RDW
• MCH (mean corpuscular hemoglobin)
HB/HCT = 27-32 pg
• RDW (red cell distribution width)
• It is correlates with the degree of
anisocytosis
_ Normal range from 10-15%
 The Reticulocyte Count
• This important value is needed in the
evaluation of any anemia.
• Normal range 1-2%
• Retic count goes up with
– Hemolytic anemia
• Retic goes down with
– Nutritional de ciencies
_ Diseases of the bone marrow itself
Reticulocytes

Supravital Leishman’s
 Anaemia
 Decrease in the number of circulating red
blood cell mass and there by O2 carrying
capacity
 Almost always a secondary disorder

Types of
Acute versus chronic
Anaemia
Causes of Anaemia
1. Decreased production of Red Cells
- Hypoproliferative, marrow failure
2. Increased destruction of Red Cells
- Hemolysis (decreased survival of RBC)
3. Loss of Red Cells due to bleeding
- Acute / chronic blood loss (hemorrhagic)
Anaemia
Hb% < 12, Hct < 38%

Hypoproliferative Hemolytic
Retics < 2 Retics > 2
Normal CBC
 MCV
Microcytic Normocytic Macrocytic
Iron De ciency IDA Chronic disease Megaloblastic
Chronic Infections Hemoglobinopathies anemias
Thalassemias Primary marrow Liver disease/
Hemoglobinopathi disorders alcohol
es Combined Hemoglobinopathi
Sideroblastic de ciencies es
Anemia Increased Metabolic
destruction disorders
Marrow disorders
Increased
destruction
Microcytic Hypochromic
Iron related tests Normal IDA
Serum Ferritin (pmo/ 33-270 < 33
L)
TIBC (µg/dL) 300-340 > 400
Serum Iron (µg/dL) 50-150 < 30
Saturation % 30-50 < 10
Bone marrow Iron ++ Absent
 Microcytic MCV < 80 , RBC < 6 µ
 RDW Widened with low MCV
 Hypochromic MCH < 27 pg, MCHC < 30%
 RI <2
 Serum ferritin Very low < 30 (p mols/L)
 TIBC Increased > 400 (µg/dL)
 Serum Iron Very low < 30 (µg/dL)
 BM Fe Stain Absent Fe
 Response to Fe Rx. Excellent
Microcytic Anaemias
MCV < 80 Serum TIBC BM Perls stain
Iron
Iron Def. Anemia ↓↓ ↑↑ 0
Chronic Infection ↓↓ ↓↓ ++
Thalassemia ↑↑ N ++++
Hemoglobinopathy N N ++
Lead poisoning N N ++
Sideroblastic ↑↑ N ++++
Macrocytic Anaemias
A. Megaloblastic Macrocytic – B12 and Folate↓
B. Non Megaloblastic Macrocytic Anaemias
1. Liver disease/alcohol
2. Hemoglobinopathies
3. Metabolic disorders, Hypothyroidism
4. Myelodystrophy, BM in ltration
5. Accelerated Erythropoesis - ↑destruction
6. Drugs (cytotoxics, immunosuppressants, AZT,
anticonvulsants)
Megaloblastic Anemia
Macrocytosis
Anaemia of Chronic Disease
 Thyroid diseases • IBD
 Malignancy – Ulcerative Colitis
 Collagen Vascular – Crohn’s Disease
Disease • Chronic Infections
 Rheumatoid Arthritis – HIV, Osteomyelitis
 SLE
 Polymyositis
– Tuberculosis
 Polyarteritis Nodosa
• Renal Failure
‘Dimorphic’ Anaemia
 Folate & Fe de ciency (pregnancy, alcoholism)
 B12 & Fe de ciency (PA with atrophic gastritis)
 Thalassemia minor & B12 or folate de ciency
 Fe de ciency & hemolysis (prosthetic valve)
 Folate de ciency & hemolysis (Hb SS disease)
 Peripheral smear exam is critical to assess these
 RDW is increased very much
RBC Size – Anisocytosis
Di erent sizes of RBC
Poikilocytosis
Di erent Shapes of RBC
Polychromasia - Spherocytosis
Target Cells

1. Liver Disease
2. Thalassemia
3. Hb D Disease
4. Post
splenectomy
 WBC
• WBCs are involved in the immune response.
• The normal range: 4 – 11x10^9 /L
• Two types of WBC:
1) Granulocytes consist of:
– Neutrophils: 50 - 70%
– Eosinophils: 1 - 5%
– Basophils: up to 1%
2) Agranulocytes consist of:
- Lymphocytes: 20 - 40%
– Monocytes: 1 - 6%
 WBC
The type of cell a ected depends upon its primary
function:
In bacterial infections, neutrophils are most
commonly a ected
In viral infections, lymphocytes are most commonly
a ected
In parasitic infections, eosinophils are most
commonly a ected.
 Neutrophil
• polymorphneuclear leukocytes (PMN,s)
• Nucleus 3-5 lobes.
• Diameter 10-14 µm
• 50-70% WBC
=2.5-7.5x10^9/ L
• Function: Phagocytosis of bacteria and cell
debris
• Numbers rise with all manner of stress,
especially bacterial infections
Neutrophil disorders
– Neutrophilia – an increase in neutrophils
– Conditions associated with neutrophilia are:
1-Bacterial infections (most common cause)
2-Tissue destruction
e.g. tissue infarctions, burns.
3- leukemoid reaction
4-Leukemia
– Neutropenia – this may result from
1-Decreased bone marrow production
e.g. BM hypoplasia.
2-Ine ective bone marrow production
– E.g. megaloblastic anemias and
myelodysplastic syndromes.
3- post acute infection
_ e.g. typhoid fever, brucellosis.
 Eosinophil
• Bilobed nucleus
• 1-5% of WBC
=0.04-0.4x10^9/L
• Diameter about 10-14 µm
• Function: Involved in allergy, parasitic infections
• Contains: eosinophilic granules
– Eosinophilia may be found in
• Parasitic infections
• Allergic conditions and
hypersensitivity reaction
 Lymphocyte
• No speci c granules
• 20-40% of WBC
=1.55-3.5x10^9/ L
• Diameter 8-10 µm
• T cells: cellular
• (for viral infections)
• B cells: humoral (antibody)
• Natural Killer Cells
• Lymphocytosis – may indicate
_ Viral infection
e.g. Infectious mononucleosis, CMV or pertussis.
_ Bacterial infection
e.g. TB
• Lymphopenia – caused by
_Stress.
_Steroid therapy
_ Irradiation
 Abnormal resul t
• (Leukocytosis) may indicate:
of WBC
_ Infectious diseases
_In ammatory disease (such as rheumatoid arthritis
or allergy)
_Leukemia
_Severe emotional or physical stress
_Tissue damage (e.g. necrosis,or burns)
• (Leukopenia) may result from:
_ Decreased WBC production from BM.
_ Irradiation.
_ Exposure to chemical or drugs.
 Platelets
•Small granular non-nucleated discs.
•Diameter about 2-4 µm
•Normal range; 150-300x10^9 /L
•Destroyed by macrophage cells in the spleen.
•Function; involved in coagulation and blood
haemostasis.
•Life span 7-10 days
 Platelets
• Numbers of platelets
– Increased (Thrombocythemia)
• Pregnancy.
• Exercise.
• High attitudes.
• splenectomy
– Decreased (Thrombocytopenia)
• Menstruation.
• Haemorrhage.
• Bone marrow destruction or suppression e.g. leukemia
 Manifestaton of thrombocytopenia
• Petechial hemorhage.
• Easy bruising.
• Mucosal bleeding
e.g. _ epistaxes.
_ gum bleeding
Erythrocyte Sedimentation Rate
(ESR)
The erythrocyte sedimentation rate (ESR)
nonspeci c measurement used to detect and
is a
monitor an in ammatory response to tissue injury
(an acute phase) in which there is a change in the
plasma concentration of several proteins (termed
acute phase proteins).
 very simply,
 allowing a speci c amount of blood to sit in a
vertical position for a period of time (usually
one hour).
 The distance, in millimeters, that the red cells
fall during this time period is the erythrocyte
sedimentation rate and is reported in mm/hr.
 Three factors: erythrocytes, plasma
composition, and mechanical/technical factors.
I. Erythrocytes
 A factor of chief importance in determining the distance
the RBCs fall is the size or mass of the falling particle. The
larger the particle, the faster its rate of fall.
 In normal blood, the RBCs remain more or less separated.
They are negatively charged and, therefore repel each
other.
 In certain diseases, however, plasma protein
concentration may be altered, causing a reduction in the
negative charge of the RBCs and consequent formation of
rouleaux. This leads to a larger mass and an increased
sedimentation velocity.
 Macrocytes tend to settle more rapidly than microcytes.
RBCs show an alteration in shape, such as sickle cells and
spherocytes, are unable to aggregate or form rouleaux
and the sedimentation rate is decreased.

  Anisocytosis and poikilocytosis reduce the ability of
RBCs to form large aggregates and there by tend
to falsely lower ESR.
 In severe anemia, the ESR is markedly elevated: the
concentration of RBCs is decreased, aggregation
and rouleaux formation are increased, and they
therefore, settle out more easily and rapidly.
 In polycythemia, in which the RBCs count is high,
the ESR is generally normal.

 Normal values:
 Adult male 0-15 mm/hr
 Adult female 0-20 mm/hr
 II. Plasma composition
 The plasma composition is the most important factor
determining the ESR. Rouleaux and aggregation of the
RBCs are controlled primarily by the levels of acute phase
proteins (most notable brinogen, -1 globulin, and -2
globulin); increasing these three plasma protein levels are
increased in the plasma.
 As the concentration of protein increases so does the
viscosity of the plasma. Although an increased plasma
viscosity will tend to inhibit the fall of the RBCs, the
increase in plasma proteins are generally those which
cause rouleaux and aggregation of the RBCs, which
a ects the ESR more greatly than does the increased
plasma viscosity. Increased concentrations of albumin will
tend to lower the ESR.
 III. Mechanical/Technical
factors
 It is important that the ESR tube be exactly perpendicular.
A tilt of 30 can cause errors up to 30%.
 Also, the rack holding the tubes should not be subject to
any movement or vibration.
 Minor, everyday variations in room temperature do not
signi cantly a ect the ESR. With large changes in
temperature, however, the sedimentation rate increases
as the temperature increases.
 The length and inner diameter of the ESR tube also a ect
the nal test results. ESR tubes with a narrower than
standard bore will generally yield lower sedimentation
rates.
 An elevated ESR may be found in:
1 Pregnancy (after the third month).
2 Acute and chronic infections.
3 Rheumatic fever.
4 Rheumatoid arthritis.
5 Myocardial infection.
6 Nephrosis.
7 Acute hepatitis.
8 Menstruation.
9 Tuberculosis.
10 Hypothyroidism.
11 Hyperthyroidism.
 Adults over 60 years of age frequently have a
slightly higher ESR value due primarily to
decreased concentrations of plasma albumin.
 A decreased ESR will be present in:
1 Polycythemia.
2 Congestive heart failure.
3 Hypo brinogenemia.
4 The presence of red blood cell abnormalities
(poikilocytosis, spherocytes, and sickle cells).
Westergren method
 Reagents and Equipment:
1) Westergren pipette calibrated in
millimeters. The National Committee for
clinical laboratory standards has set
speci c dimensions for the pipettes
2) Westergren rack.
3) Disposable pipettes
4) Leveling plate for
holding the Westergren
rack
5) Timer.
 Specimen
 Whole blood (4 volumes) diluted with 0.109
M trisodium citrate (1 volume). Alternatively,
3ml whole blood anticoagulated with EDTA.
(If this specimen type is used the whole
blood [2.0ml] must be diluted with 0.85% w/
v sodium chloride [0.5ml] prior testing).
Wintrobe and Landsberg method
 Reagent and equipment:
 Wintrobe tube, calibrated in millimeters
 Wintrobe pipette rack.
 Disposable capillary pipette.
 Applicator sticks
 Specimen:
 Whole blood, 1 ml, using EDTA as the anticoagulant.
The ABO System
 Discovered in 1901 by Dr. Karl Landsteiner
 4 Main Phenotypes (A, B, AB, O)
 ABO gene located on long arm of
chromosome 9
ABO Antibodies
 2 glycolipid isoantigens called A and B found
on the surface of RBCs
 display only antigen A -- Blood Type A
 display only antigen B -- Blood Type B
 display both antigens A & B -- Blood Type AB
 display neither antigen -- Blood Type O
 Plasma contains isoantibodies or agglutinins
to the A or B antigens not found in blood
 Anti-A antibody reacts with antigen A
 Anti-B antibody reacts with antigen B
Antigens & Antibodies
Blood Group Antigens on Antibodies in Serum Genotypes
RBCs

A A Anti-B AA or AO

B B Anti-A BB or BO

AB A and B Neither AB

O Neither Anti-A and anti-B OO

ABO & Rh(D) 55

Signi cance of ABO Group
 ABO mismatched transfusions:
 Rare
 May be life threatening
 Can be caused by technical or clerical error
 Intravascular haemolysis
 More severe in group O patients

ABO & Rh(D) 57

 Universal Donor and Recipient
 Universal Donor  Universal Recipient
 Group O  Group AB
 Carries no A or B  Patient has no anti-A
antigens or anti-B present
 Packed and processed  Cannot lyse any
units have little transfused cells
antibody  antibodies may be
present
The Rh(D) Antigen
 RH is the most complex system, with over
45 antigens
 Discovered in 1940 after work on Rhesus
monkeys
 Subsequently discovered to be unrelated
to monkeys
 RH gene located on short arm of
chromosome 1
 Rh(D)
 86% of population are Rh(D) positive
 The d gene is recessive:
Dd, dD, DD, pos

 Only dd, neg
 Anti-D can also be stimulated by
pregnancy with an Rh(D) +ve baby & Rh –ve
mother
 Sensitisation can be prevented by the use of anti-
D immunoglobulin, antenatally and post natally
 Rh(D) -ve females of childbearing potential
should never be given Rh(D) +ve blood
products